This project involves the physical and genetic characterization of a complex group of closed-circular DNA plasmids in the Gram-positive bacterium Bacillus megaterium. These plasmid molecules, which are distributed among 8 to 9 discrete size classes, are being examined by the techniques of DNA-DNA hybridization, restriction endonuclease cleavage, renaturation kinetic analysis, and by electron microscopy. The combination of these approaches has shown that, for the two smallest species (6 kb and 9.2 kb, respectively) the molecules within each size class are for the most part unique in their nucleotide sequences. The possible existence of a fraction of slow renaturing sequences is being examined - these are thought to lie in a 1.2 kb 'silent region' of the restriction endonuclease map of the 6 kb plasmid. The mode (s) of replication and maintenance of the plasmid molecules are being approached through the study of temperature-sensitive replication and drug-resistant mutants, and by attempts to transfer the plasmids to other bacterial species by transformation, using congruent selection. The smaller plasmid species will also be cloned by recombinant DNA techniques to study their behavior in foreign cytoplasms.